THE BIOSYNTHESIS ANDTURNOVEROF OXALATE IN NORMALANDHYPEROXALURICSUBJECTS*

By T. DAVID ELDERAND JAMES B. WYNGAARDEN

(From the Department of Medicine, Duke University Medical Center, Durham, N. C.)

(Submitted for publication February 10, 1960; accepted March 31, 1960)

Oxalate is a normal constituent of human urine.Since oxalate is found in many foods it has fre-quently been assumed that urinary oxalate islargely, if not entirely, of dietary origin. How-ever, animal studies have shown that oxalate mayarise endogenously, and glycine, glyoxylic acid,glycolic acid (3-5) and ascorbic acid (6, 7) havebeen shown to be precursors of urinary or tissueoxalate under various circumstances.

The origin of urinary oxalate has long been animportant physiological problem because of thehigh incidence of calcium oxalate urolithiasis inmany parts of the world. Recently, interest in thebiosynthesis of oxalate in man has been greatlystimulated by recognition of the syndrome of pri-mary hyperoxaluria and oxalosis, a disorder inwhich excessive production of oxalate occurs andleads to deposition of calcium oxalate crystals inrenal parenchyma and extrarenal tissues. Ascor-bic acid has previously been shown to be a pre-cursor of some urinary oxalate of normal man(6, 8). In this paper glycine is shown to be amajor precursor of urinary oxalate of normal andhyperoxaluric subjects. In one subject with pri-mary hyperoxaluria, glycine incorporation intourinary oxalate was excessive, whereas in anothersubject with an inconstant hyperoxaluria, glycineincorporation was not abnormal. Measurementsof the miscible pool of oxalate and of the rate ofits turnover have been made by conventional iso-topic dilution techniques, and a gross expansion ofthe miscible pool of oxalate demonstrated in ahyperoxaluric subject.

EXPERIMENTALMETHODS

Materials. Glycine I-C14, specific activity 2.2 mc permmole, and oxalic acid 1,2-C"4, specific activity 2.6 mcper mmole, both of radiopurity > 99 per cent, were pur-chased from Isotopes Specialties Co. Burbank, Calif.

Control subjects. Control subj ects were hospitalized* Supported in part by a grant (A-1391) from the

United States Public Health Service. Preliminary re-ports of this work have appeared (1, 2).

patients without known metabolic, gastrointestinal orgenitourinary disease, in whom routine tests [urinalysis,blood urea nitrogen (BUN), phenolsulfonphthalein(PSP) excretion] disclosed no evidence of renal disease.They were permitted a regular hospital diet except thatfoods of high oxalate content [cocoa, chocolate, tea, rhu-barb, beets and spinach (9)] were withheld for a fewdays before and during the study period. The basal ex-cretion of oxalate [as (COOH),-2H,O] ranged from11.6 to 36.0 mg per day in the control subjects.

Hyperoxaluric subjects. C.B. was a 30 year old malewho was first seen at Duke Hospital in 1950 at the age of23. He had had recurrent urolithiasis since age 10 andhad required pyelolithotomy at ages 18 and 19. On ad-mission in 1950 nephrolithotomy and pyelolithotomy wereagain performed, urinary tract infection was present andthe stones were found to contain calcium, oxalate, phos-phate and small amounts of carbonate. Subsequent to1950 he required litholapaxy on several occasions. Hepassed gravel almost daily, and had severe renal colicon numerous occasions each month. Repeated analysesof serum calcium, phosphorus and alkaline phosphatasewere normal. Mild elevations of nonprotein nitrogen(NPN) (maximum, 60 mg per 100 ml), serum uric acid(maximum, 6.2 mg per 100 ml) and cholesterol (268 mgper 100 ml) were noted, and PSP excretion tests rangedfrom 41 to 45 per cent in 2 hours. Urine cultures re-vealed organisms of the Klebsiella and Aerobacter groups,and he was treated with various agents without success.

In 1958 he was found to be excreting abnormal quan-tities of oxalate, ranging from 103 to 143 mg per day.He was therefore admitted for further study. Generalphysical examination was normal except for well healedsurgical scars. Blood pressure was 114/80. Urinalysisrevealed pH 6.5, specific gravity 1.011, a trace of protein,and innumerable leukocytes in the sediment. NPN was48, serum uric acid 6.8, calcium 9.3, and phosphorus 3.5mg per 100 ml. X-ray revealed bilateral staghorn calculibut no evidence of nephrocalcinosis. He was placed ona diet calculated to contain 62 mg of oxalate daily dur-ing the study period.

A familial history of urolithiasis was obtained, and asister was found also to have a slightly elevated urinaryexcretion of oxalate (65 mg per day). This sister haspassed stones composed of calcium oxalate; both parentsand a paternal cousin have also passed stones. Theparents and a brother of the patient died of nonrenaldisorders.

N.M., a 48 year old female with a 25 year history ofrecurrent calcium oxalate urolithiasis, was studied 4

1337

T. DAVID ELDER AND JAMES B. WYNGAARDEN

months following left nephrectomy. The kidney was non-functioning and showed chronic pyelonephritis and hy-dronephrosis but no nephrocalcinosis or parenchymaloxalate deposits. One week after operation her urinaryoxalate excretion was 117 mg per 24 hours; 4 monthslater when she was admitted for further studies it wasinitially 90 mg per 24 hours. She was placed on a stand-ard diet calculated to contain each day: protein 30.4 g,calcium 112.5 mg, ascorbic acid 33 mg, pyridoxine 0.5mg. By analysis it contained 129 mg of oxalic acid di-hydrate per day. After 6 days on this diet her urinarycalcium excretion was 400 to 600 mg per day. Serumcalcium was 9.1 mg per 100 ml, phosphorus 4.2 mg per100 ml and alkaline phosphatase 2.6 U per 100 ml.Studies to date have not established the cause of herhypercalcuria. Because of a decline in hyperoxaluriawhile on the experimental diet, she remained on a 30 gprotein regimen following the study. 'Urinary oxalateexcretion has remained below 40 mg per day on 3 differ-ent examinations over a 5-month period. Her urine sedi-ment is now free from calcium oxalate crystals, whichwere formerly present in abundance in all specimens.

Tracer studies. Glycine 1-C'4 was administered orallyto subjects in the fasting state in 25 or 50 Ac doses (11.4or 22.7 ,g) in 100 ml of water. Oxalic acid 1,2-C4 wasadministered slowly intravenously in 1 ,uc doses (0.05 mg)in 2 ml of sterile (Seitz-filtered) solution in water. Bothcompounds were administered after an overnight fast.

In two subjects oxalate and hippurate were isolatedsimultaneously, from successive 2-hour urine samples.Two hours after ingestion of labeled glycine, and at 4-hour intervals for 3 additional doses, the subjects ingested3 g of sodium benzoate, so as to insure a large urinaryoutput of hippuric acid.

Urine specimens were collected in clean glass jugscontaining 5 ml of concentrated HCI. Respiratory CO.was collected in carbonate-free, 1 N sodium hydroxideand precipitated as barium carbonate.

All counting was done in stainless steel planchets in aRobinson gas-flow counter (10). Calcium oxalate sam-ples varied in mass from 4 to 36 mg and were correctedfor self-absorption to a standard mass of 22 mg perplanchet (1.54 sq cm). Barium carbonate samples werecounted in infinite thickness. Hippuric acid samplesweighed from 5 to 15 mg and were corrected for self-ab-sorption to a standard mass of 3.3 mg per planchet. Ap-propriate conversion factors were employed for compari-son of counting values of glycine, oxalic acid, calciumoxalate and hippuric acid when required.

Isolation of oxalate from ulrine. Each urine samplewas filtered through Whatman no. 42 paper in a Buchnerfunnel if any cloudiness or precipitate was grossly visible,and aliquots were taken for determination of oxalate bythe method of Archer, Dormer, Scowen and Watts (11).Thirty mg of oxalic acid dihydrate was added to theremainder of each urine specimen as carrier and the vol-ume reduced to approximately 20 ml under reduced pres-sure at 30' C. The pH was adjusted to 5.5 with 8 Nammonium hydroxide, and 5 ml of 5 per cent CaCl, wasthen added. The samples were stored at 40 C overnight,centrifuged, and the supernatant solution discarded.

Calcium oxalate was then purified by a slight modifica-tion of the method of Burns, Burch and King (12). Theprecipitate was washed 2 or 3 times with 4 ml of 0.35N NH4OH, to remove colored impurities, and dissolvedin 6 ml of 1 N sulfuric acid; after centrifugation theresidue was discarded. The supernatant was placed in aglass microextractor 1 and extracted continuously with100 ml of ether for at least 6 hours. At the end of theextraction period 10 ml of distilled water was added tothe flask and the ether evaporated on a steam bath. Theaqueous phase was transferred to a 40 ml conical cen-trifuge tube and the pH was adjusted to 5.5 with 8 Nammonium hydroxide. Calcium oxalate was reprecipi-tated by addition of 5 ml of 5 per cent CaCl2. The sam-ple was stored overnight in the cold, centrifuged, and thesupernatant discarded. The precipitate was dissolved in1 N HCl, treated with Darco, filtered, and recrystallizedby adjustment of the pH to 5.5 and addition of 5 ml of5 per cent CaCl,. The last procedure was repeated sev-eral times.

The samples were dried at 80' C in a vacuum oven,ground with a stout glass rod in acetone, and transferredby pipet to a tared stainless steel planchet for counting.

Isolation of hippuric acid fromii urine. Urinary hip-puric acid was isolated according to Quick (13) andpurified by treatment with Darco and by repeated re-crystallizations from hot water (14). The melting pointsof the isolated samples were identical with those of au-thentic hippuric acid.

Calculationts. The standard isotopic dilution equationfor calculating the miscible pool of oxalate is as follows(15) : A = a (It/Io - 1), where A is the size of the mis-cible pool, a is the quantity of labeled material added tothe pool, Ii is the specific activity of the injected material,and 1. is the theoretical specific activity in the misciblepool at the moment of mixing. In the present study thisequation was simplified, and the pool was calculated asfollows: A = (ali)'/Io. In this equation (aIr)' repre-sents the total counts administered minus the labeledoxalate which was excreted too rapidly to have mixedthoroughly within the pool (see below). The term"-1" of the first equation may be ignored, since "a" was

very small (0.05 mg) compared with values of A (4 to8 mg). The intercept, I, and the slope, - K, and theirstandard deviations, were calculated by the method ofleast squares. The biological half-time, T!, was obtainiedfrom the expression (15) K= log 2/Ti. The standarddeviation of A was calculated as previously described(16).

RESULTS

Oxalate excretion

The urinary excretion of oxalate in 18 normalsubjects (37 samples) on a mixed diet ranged from6.3 to 44.8 mg, and averaged 22.1 mgper 24 hours,[as oxalic acid dihydrate, (COOH) , 2H,O1.No consistent diurnal variation was found in two

1 Ace Glass Co., Inc., Vineland, N. J., Catalog no. 6820.

1338

OXALATE BIOSYNTHESIS ANDTURNOVER13

I ~~~~~~~~~~~ADMc.1E di

E 41 L.OXALATENCONTRL ANDHYERXhRLcollections wereobtained,nHyperoxolurics

A coe(x1 = 0 0 0.2

0

0 4 8 12 16 20 24 28 32 36HOURS

FIG. 1. INCORPORATIONOF GLYCINE 1-C14 INTO URINARY

OXALATE IN CONTROLAND HYPEROXALURICSUBJECTs Thedata have been normalized to a standard dose of 25nCofC14 given orally as carrier-free glycine 1- C14.

subjects in each of whom 12 consecutive 8-hourcollections were obtained, nor was urinary oxalateexcretion significantly correlated with urine vol-ume (R = 0.87, 0.68; p = 0.06, 0.20).

Glycine incorporationUrinary oxalate became highly labeled within a

few hours following oral administration of 25 /Acof glycine 1-C'4 in each of six control subjects(Figure 1). The initial (3-hour) specific activityvalues ranged from 49,000 to 450,000 cpm permmole. This ninefold range was found in sub-jects having only a threefold range in urinary

HOURS

FIG. 2. CUMULATIVE INCORPORATIONOF GLYCINE 1-C"INTO URINARY OXALATE IN CONTROLAND HYPEROXALURIC

SUBJECTS. Data are expressecld . percentage of adminis-tered C".

HOURS

FIG. 3. COMPARISONOF ENRICHMENTOF URINARY OX-ALATE AND HIPPURATE IN TWOCONTROLSUBJECTS FOLLOW-

ING INGESTION OF 25 JAC OF GLYCINE 1-C". Sodium ben-zoate, 3 g, was given orally at 2, 6, 10 and 14 hours toinsure a large urinary output of hippurate.

oxalate values; specific activity values were notinversely correlated with excretion values. En-richment values declined rapidly so that, by 24 to30 hours, the counting values of urinary oxalateapproached background.

The cumulative incorporation of C14 into urinaryoxalate (Figure 2) ranged from 0.027 to 0.081per cent (mean, 0.051 per cent) of the adminis-tered dose of glycine 1-C14 in five control subjects.

In two control subjects who received load dosesof sodium benzoate beginning 2 hours after in-gestion of glycine (Figure 3), urinary oxalatewas significantly more highly labeled than was uri-nary hippurate between 2 and 10 hours. Bothsubstances exhibited typical die-away enrichmentcurves in both subjects.

These studies establish that glycine serves as aprecursor of at least some of the urinary oxalate innormal man, and suggest 1) that relatively littledilution occurs between glycine and oxalate, 2) thatthe miscible pool of oxalate is small, and 3) thatthe turnover of the oxalate pool is rapid. Meas-urements of the miscible pool of oxalate and of itsturnover were then attempted by standard isotopicdilution techniques.

The miscible pool of oxalate and the rate of itsturnover

When 50 ,ug of oxalic acid 1,2-C14 was injectedintravenously into control subjects, a portion rap-

1339

T. DAVID ELDER AND JAMES B. WYNGAARDEN

idly appeared in urine. A semilogarithmic plotof specific activity of turinary oxalate against timeshowed that a linear decay wvas achieved after 4hours (Figures 4, 5). After about 18 hoturs thespecific activity values were so low that their ac-curacy was doubtful, and their usefulness small inthis study.

By the time that mixing of the labeled oxalatewithin the oxalate pool was complete, presumablyabout 4 hours after its administration, a large frac-tion of the test dose of labeled oxalate had alreadybeen excreted. Since eventually 89 to 99 per centof the administered tracer was recovered un-changed in urine (see below), nonrenal loss of oxa-late was not an appreciable factor, and a correctionfor the labeled oxalate which had failed to mixadequately could be applied on the basis of the 0 to4 hour urinary fraction. The regression of thelinear portion of the specific activity-time curvewas calculated by the method of least squares,omitting data of 0 to 2 and 2 to 4 hour points. Ineach case the zero intercept was a highly sig-nificant value, p < 0.01. The oxalate-C14 ex-creted in excess of that anticipated from the ex-trapolated curve was then subtracted from the ad-ministered dose (ali) and further calculationswere made on the basis of the "corrected adminis-tered dose" (ali)'. The subtrahend averaged

100,00

o G.R.

10,000

~~0~~

E 1000-t

100 _

10 x0 3 6 9 12 I5 l8 21

HOURS

FIG. 4. SPECIFIC ACTIVITY OF URINARY OXALATE FOL-LOWING INTRAVENOUS ADMINISTRATION OF OXALIC ACID1,2-C14 TO A CONTROLSUBJECT. The data of this experi-ment (Subject G.R., Table I), when analyzed by themethod of least squares, indicated a miscible pool of 4.9 +0.7 mg, and a turnover rate of 1.4 ± 0.3 mg per hour.

3,000O

1000o

300

look

30

-o 4 8 12 16HOURS

20 24 28

FIG. 5. SPECIFIC ACTIVITY OF URINARY OXALATE FOL-

LOWING INTRAVENOUS ADMINISTRATION OF OXALIC ACID

1,2-C1 TO A HYPEROXALURICSUBJECT. These data (Sub-ject N. M., Table I) indicated a miscible pool of 39.1 +2.4 mg and a turnover rate of 4.6 0.3 mg per hour.

about 60 per cent of (aIi), and therefore the meas-

urements of oxalate and of specific activity in theinitial samples are critical to this correction.

Table I shows that the calculated miscible poolof oxalate (expressed as oxalic acid dihydrate)ranged from 4.9 + 0.7 to 8.6 1.1 mg in threecontrol subjects. The biological half-time of thepool ranged from 2.2 + 0.3 to 2.8 0.3 hours.The calculated daily turnover of oxalate in thesethree control subjects ranged from 34.1 + 6.7 to63.8 + 11.3 mg per day. In each case the valueof urinary oxalate on the day of the study fellwithin 2 SD of the calculated turnover.

Physiological disposition of oxalate in man

In three studies on control subjects and in twostudies on N.M., a hyperoxaluric subject (see be-low), from 89 to 99 per cent of the injected oxalate1,2-C14 was recovered unchanged in urine in 36hours.

Respiratory CO, was collected at frequent in-tervals for 6 hours from two control subjects whohad received 1 ,tc of oxalate-C14 intravenously andorally, respectively. In both cases all samples ofCO2, counted as barium carbonate, were devoid

0

N.M.

0

~-o

I

Io

tl/2

\ _

l l l~~~

1340

OXALATEBIOSYNTHESIS AND TURNOVER

of enrichment by the counting method employed,0>.=,, go>c>o which would have detected as little as 0.01 per

cent of the administered dose in any single sample.

I. enc6*, 46O -

Studies on patients with hyperoxaluria and recur-rent calcium oxalate urolithiasis

| I -°O"r° I Subject C. B. The male subject with probablex -w -H -H --H-H primary hyperoxaluria was given an oral dose of

c 4o° ccX 4 25 uc glycine 1-C14, and incorporation of C14 intourinary oxalate was demonstrated. The specificactivity values were comparable with those found

> C;C;66666 in control subjects at equivalent points in timeiz<EH H H -H -Htl (Figure 1); however, a cumulative incorporation

i 4 C6 of 0.22 per cent of the administered dose was

found (Figure 2), a value some fourfold greater, 66666C; than the mean cumulative incorporation value

-H -H -H -H -H found in the control subjects.tn0

- -4\6666o Subject N.M. During the first week on the

special diet the urinary oxalate excretion of N.M.,the female hyperoxaluric subject, progressively

HC'

-C;C; declined, and after the seventh day it remained

."i cli cli\C;o .4 within the normal range. The factors involved

in the disappearance of hyperoxaluria in this pa-- -'_ o tient are not understood and are the subject of

<XHIHoHH66 continuing studies which will be reported else->6.4 06OO+ Oo _where. The studies with glycine 1-C and oxalic

acid, 1,2-C'4 will be of interest to record here.Xe Oo Following oral administration of 25 ,c of gly-

cine 1-C'4, her urinary oxalate was maximally la-; -H 1-H -H- H beled in the initial (0 to 2 hour) specimen. The

specific activity values were similar to those foundin E.L., who showed the lowest enrichment of the

*_e ^ <, six control subjects (Figure 1). After 30 hours,o

°+ t n n Oo urinary oxalate contained only trivial quantities-H - -H -Hi -H of isotope. The cumulative incorporation of C'4

. e e 00 t OO into urinary oxalate was 0.047 per cent, a valueX.11̂ 0\^ *=,4 close to the mean of those obtained in the con-

trol subjects (Figure 2).1 C)000o + .'t The miscible pool and the rate of its turnover,._§+^+^o,,were determined twice on this patient. On the

E second day of the study her pool was 39.1 + 2.4;=. ° mgand its turnover 109.7 ± 6.7 mg per day (Fig-

14! 00o°oxor- \0 \ ure 5). On this day her urinary oxalate excre-:rt10tion was 80.6 mg per 24 hours. Subsequently,

be Oo0 - 00c when her hyperoxaluria had disappeared, her mis-cible pool was found to be 1.8 + 1.0 mg and its

. ( /)turnover 8.2 ± 3.8 mg per day. Her urinary oxa-:sJVxzz* § late excretion on the day of this study (Day 14)

was 12.3 mg per 24 hours.

1341

T. DAVID ELDERANDJAMES B. WYNGAARDEN

DISCUSSION

Physiological disposition of oxalate. Dakin(17) suggested in 1922 that oxalate is a nones-

sential end product not further metabolized inmammalian tissue. Subsequent studies in animals(4, 18, 19) and the present studies in man haveshown that labeled oxalate is not significantly con-

verted to respiratory CO2, and that an intravenoustracer of labeled oxalate is almost quantitatively ex-

creted in urine.Biosynthesis of oxalate. Ratner, Nocita and

Green (3), Weinhouse and Friedmann (4), andNakada and Weinhouse (5) have shown in animalstudies in vivo and in vitro that glycine may beconverted to oxalic acid, via glyoxylic acid. Gly-cine is converted to glyoxylic acid by glycine oxi-dase, and possibly as a by-product of transamina-tion with a-ketoglutaric acid, a reaction whoseequilibrium, however, lies far toward glycine.Glyoxylic acid is converted to oxalic acid by en-

zymes which are less well understood. One may

be xanthine (aldehyde) oxidase (20) ; another,possibly a mutase, also exists (3). Glyoxylicacid is the only known direct precursor of oxalicacid in mammalian systems. Glycolic acid isreadily converted to glyoxylic acid by a flavopro-tein, glycolic oxidase (21), and sparingly by lac-tic dehydrogenase and DPN (5). The pathwayby which ascorbic acid (4, 16) contributes carbonatoms to oxalic acid is not known. In bacterialsystems (22), oxalosuccinic acid, bxaloacetic acidand fl-ketoadipic acid also serve as precursors ofoxalic acid.

In the control subjects, 0.03 to 0.08 per cent ofglycine 1-C14 was converted to oxalic acid. Thenormal urinary excretion of oxalic acid (mean 22mg) approximates 0.03 to 0.08 per cent of theturnover of the first metabolic glycine pool in man,

estimated to be about 0.5 to 1 g per kg per day(23, 24), or 35 to 70 g per 70 kg man per day.Thus, glycine is at least a major precursor of uri-nary oxalate in normal persons, but the apparentcorrespondence of values given above should notbe stressed unduly, for there is also evidence thatan appreciable percentage of urinary oxalatecomes from precursors other than glycine. Craw-hall, Scowen, de Mowbray and Watts (25) stud-ied a single control subject with glycine 1-C13,and concluded that at least half of the urinary

oxalate was not derived from glycine in this sub-ject.

In the present studies urinary oxalate was la-beled considerably in excess of urinary hippurate.When no sodium benzoate is administered, uri-nary hippurate and urinary free glycine are com-parably labeled within a few hours followingadministration of glycine N'5 or C13 (25). Thework of Gray and Neuberger (26) and of Arn-stein and Neuberger (27) suggests that large dosesof benzoate stimulate synthesis de novo of newglycine, or mobilize glycine from pools not ordi-narily in rapid equilibrium with the pool fromwhich hippurate derives its glycine. Benzoateloading probably explains why hippurate glycinewas less highly labeled than oxalate in the pres-ent studies, whereas in the studies of Crawhall andassociates (25) free glycine (and presumably hip-purate also) was more highly labeled than wasoxalate. Finally, the studies reported herein of-fer additional evidence for the existence of mul-tiple metabolic pools of glycine in poor equilibriumone with another, as previously suggested by stud-ies of a number of other workers (14, 27, 28).

The miscible pool of oxalate and its biologicalturnover. The isotopic dilution technique for de-termination of the pool and turnover of a sub-stance is based upon a number of assumptions andis subject to limitations which have been discussedin detail previously (29). In the present studythe major limitations are two: 1) the relativelylarge fraction of the injected tracer which is rap-idly excreted in urine, and 2) the small quantityof oxalate present in 2-or 4-hour urine specimens.The first problem has been approached by elimi-nating from calculations the labeled oxalate whichwas excreted without mixing with the pool. Thesecond problem was minimized by employing theaverage of replicate analyses.

The values shown in Table I for the misciblepool, biological half-time and turnover of oxalatein the body are in reasonably close agreement inthree control subjects. The turnover values arein satisfactory agreement with urinary excretiondata, although the turnover values tended to be alittle larger, both in controls and in the hyper-oxaluric subject. Animal studies suggest thatsome endogenous oxalate is excreted in the feces,but the urinary recovery of labeled oxalate of 94per cent suggests that nonrenal excretion of en-

1342

OXALATEBIOSYNTHESIS AND TURNOVER

dogenous oxalate is not a large fraction in normalsubjects. Since only 90 per cent of oxalate C14was recovered in N.M. when her oxalate pool waslarge, whereas 99 per cent was recovered whenit was small, the possibility remains that a largerfractional nonrenal disposal of oxalate occurs inthe presence of expanded body stores of oxalate.

Hyperoxaluria. In C.B., the incorporation ofglycine 1-C14 into urinary oxalate was 0.22 percent, a value about fourfold greater than the meancontrol value. This patient's urinary oxalate ex-cretion was four- to fivefold greater than the meannormal value. Thus it appears that about thesame percentage of urinary oxalate was derivedfrom glycine in C.B. as in controls. Crawhall,Scowen and Watts (28) reached a similar con-clusion in two hyperoxaluric subjects given gly-cine 1-C13. These results, considered togetherwith evidence in vitro that oxalate synthesis fromglycine becomes appreciable only when the gly-oxylic acid pool is greatly expanded, suggest thata block in glyoxylic acid metabolism may exist inprimary hyperoxaluria, possibly of glyoxylic-glutamic transaminase (30) or of glyoxylic de-hydrogenase (31). Either block (Figure 6)could account for overproduction of oxalate frommore than one precursor of its obligatory inter-mediary, glyoxylic acid (32).

In N.M. the incorporation study was performedat a time when the miscible pool and the daily ex-cretion of oxalate were declining and approachingnormal values (Day 5). The metabolic processwhich had led to hyperoxaluria had inadvertentlybeen corrected, and the possibility that oxalate hadpreviously been overproduced from glycine is notexcluded. What relationship, if any, the hyper-oxaluria demonstrated in this subject bears tothat found in patients with primary hyperoxaluriaand oxalosis is obscure at present. Secondary

GLYCOLIC ACID

GLYCINE < GLYOXYLIC ACID *OXALIC ACID

Hypothetical 4(Sites of Block JWI

CO2 + FORMIC ACID

FIG. 6. POSSIBLE SITES OF HYPOTHETICAL BLOCK IN

PRIMARY HYPEROXALURIA.

hyperoxaluria is known to occur in man in sar-coid and hepatic cirrhosis (33) and in animalswhich are deficient in pyridoxine (34). Addi-tional studies are required to elucidate the natureof the metabolic or absorptive defect responsiblefor the inconstant hyperoxaluria in this subject.

SUMMARY

1. In five control subjects, from 0.027 to 0.081per cent of a tracer dose of glycine 1-C14 appearedin urinary oxalate, apparently by a fairly directbiosynthetic route. The sequence, glycine-*glyoxylate-> oxalate, is known in animals.

2. The miscible pool of oxalate ranged from 4.9to 8.6 mg in three control subjects; its turnoverranged from 34 to 63 mg per day. The latter val-ues agreed satisfactorily with excretion values.Labeled oxalate was not catabolized to CO,, andwas recovered almost quantitatively (89 to 99 percent) in urine.

3. In one hyperoxaluric subject, 0.22 per cent ofa glycine 1-C14 tracer appeared in urinary oxalate,a value four times the mean control value. In asecond patient, the miscible pool of oxalate de-clined from 39.1 to 1.8 mg and its turnover from109.7 to 8.2 mg per day during the study period.The nature of the metabolic defect in this patient,who incorporated glycine into oxalate normally, isobscure.

REFERENCES

1. Wyngaarden, J. B., and Verner, J. V. The biosyn-thesis of oxalate from glycine 1-C"4 in man. Clin.Res. 1958, 6, 267.

2. Elder, T. D., and Wyngaarden, J. B. The synthesisand turnover of oxalic acid in normal and hyper-oxaluric subjects (abstract). J. clin. Invest. 1959,38, 1001.

3. Ratner, S., Nocita, V., and Green, D. E. Glycineoxidase. J. biol. Chem. 1944, 152, 119.

4. Weinhouse, S., and Friedmann, B. Metabolism oflabeled 2-carbon acids in the intact rat. J. biol.Chem. 1951, 191, 707.

5. Nakada, H. I., and Weinhouse, S. Studies of gly-cine oxidation in rat tissues. Arch. Biochem. 1953,42, 257.

6. Hellman, L., and Burns, J. J. Metabolism of L-as-corbic-1-C"4 acid in man. Fed. Proc. 1955, 14, 225.

7. Burns, J. J. Biosynthesis of L-ascorbic acid; basicdefect in scurvy. Amer. J. Med. 1959, 26, 740.

8. Lamden, M. P., and Chrystowski, G. A. Urinaryoxalate excretion by man following ascorbic acidingestion. Proc. Soc. exp. Biol. (N. Y.) 1954, 85,190.

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T. DAVID ELDERANDJAMES B. WYNGAARDEN

9. Andrews, J. C., and Viser, E. T. The oxalic acidcontent of some common foods. Food Res. 1951,16, 306.

10. Robinson, C. V. Windowless, flow type proportionalcounter for counting C". Science 1950, 112, 198.

11. Archer, H. E., Dormer, A. E., Scowen, E. F., andWatts, R. W. E. Studies on the urinary excretionof oxalate by normal subjects. Clin. Sci. 1957, 16,405.

12. Burns, J. J., Burch, H. B., and King, C. G. Themetabolism of 1-C"-L-ascorbic acid in guinea pigs.J. biol. Chem. 1951, 191, 501.

13. Quick, A. J. Clinical application of hippuric acidand prothrombin tests. Amer. J. clin. Path. 1940,10, 222.

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